This invention is directed to an improved method and apparatus for regenerating whole plants from calli or tissue. In particular, the invention is directed towards a novel process and apparatus for intermittently exposing the cultivated plant tissue to a growth medium. The invention is further directed to a novel method of separating cultured plant embryos on the basis of embryo size, shape, and density through the use of an intermittent rocking action in combination with a series of separation grids, sieves, and/or baffles.
For many plant species there are well known protocols for the regeneration of whole plants from the aseptic culturing of an originally undifferentiated tissue such as protoplast, callus, or meristimatic tissue. Often, the protoplast, callus or meristimatic tissue have been genetically transformed or the meristimatic tissue is selected from a desired patented stock. The resulting protoplast, calli or tissue can then be regenerated into whole plants, including whole fertile plants that can grow in soil.
Existing methods of aseptic plant culturing involved starting with a callus or other stock material which is subcultured on a periodic basis onto a suitable fresh medium so as to induce embryo formation. As embryos are formed, they are removed and plated onto a second medium containing suitable plant growth regulators designed to bring about embryo maturation. Through periodic transfers onto fresh mediums, embryos can be differentiated into plantlets which are then transferred to another solid growth medium where root systems can be grown.
Alternative methods of plant regeneration or micropropagation involve the use of liquid cultures which immerse the various stages of the regenerating plant. While liquid culturing may be used to bring about mature viable plants, the process is expensive, prone to contamination, and requires that special steps be undertaken to successfully transfer the plants from a liquid culture environment to a condition where the plants are viable in a freestanding soil environment. In particular, the use of liquid cultures on rotary shakers results in a high shear force in the culture media. As vessel size increases, the high shear force involved in aeration creates an environment unsuitable for the growth and development of embryos and delicate organs such as leaves and shoots.
In particular, the collection and transfer of propagated embryos continues to be a labor intensive, manual process in which individual embryos are manipulated using forceps. While more mechanized solutions have been proposed, none have met with commercial success given in part the expense and complexity of the handling equipment.
It is also known in the art to propagate plant tissue into small plantlets as is taught in U.S. Pat. No. 5,597,731, incorporated herein by reference. The above patent discloses a plant propagation system in which sealed membrane vessels are used as a container for a plant material therein. The translucent vessels are permeable to gas and liquids. They are suitable for cultivating plant tissue obtained from a parent plant and which is subsequently grown heterotrophically. Once the plant material has developed the capability to photosynthesize, the sealed vessels are transferred to a greenhouse environment for photoautotrophic growth. An appropriate amount of needed gases, water, and liquid nutrient solution can be supplied in a controlled manner to the vessel trays.
Other plant propagation systems are known which use a liquid nutrient solution as set forth in U.S. Pat. No. 5,558,984, incorporated herein by reference. While a liquid nutrient solution for propagation of plant tissue offers numerous advantages in the large scale and economical propagation of plants, there remains room for improvement and variation within the art.
The present invention recognizes and addresses various disadvantages and drawbacks of prior art constructions and methods.
Accordingly, it is an object of the present invention to provide a plant micropropagation system.
It is another object of the present invention to provide a new method for propagating plant tissue.
It is a further object of the present invention to provide a micropropagation system for automatically growing plant tissues which makes use of an intermittent exposure to a liquid nutrient solution.
It is a further object of the present invention to provide a mechanized system and process for promoting the heterotrophic growth of plant tissue in an aseptic environment.
It is yet an additional object of the present invention to provide for an improved process for collecting culture-grown plant embryos.
It is a further object of the present invention to use the intermittent wave motion established within a culture vessel and in combination with a mechanical separator to separate plant embryos from non-embryonic tissues on the basis of the embryo size, shape, and density.
These and other objects are achieved by providing a mechanized micropropagation system and process in which a pivoting platform is used to periodically immerse the plant tissue maintained in a sealed vessel to the nutrient media. The pivoting movement of the platform facilitates the growth of the plant such that the majority of the time the plant is not immersed in the growth media. Furthermore, the movement of the platform engages the head of a piston-operated pumping mechanism which is used to release nutrients from a storage vessel into the plant containment vessel. As a result, the propagated plants are more suitable for release into a soil environment and the plants are cultivated in a process which limits the likelihood of widespread contamination or cross-contamination from single nutrient source.
Additional objects of the present invention are provided by a process of separating plant embryos and/or providing an embryo enriched fraction from culture media comprising the steps of separating embryos from propagated tissue cultures; providing a vessel adapted for receiving aseptic tissue and media; placing within an interior of said vessel a separation matrix consisting of an innermost layer of a substrate, a template positioned upon said substrate, said template defining a plurality of openings therethrough and providing communication with said substrate, and, a screen positioned above the substrate, said screen defining a plurality of openings; introducing into said vessel a supply of cultured tissue containing embryos and a separation medium; establishing an intermittent wave motion to said separation medium and said tissue culture; separating a plurality of individual embryos from said culture, said plurality of embryos passing through said openings of said screen and said openings of said template.
These and other features, aspects, and advantages of the present invention will become better understood with reference to the following description and appended claims.